The capacity of a commercially available gamma interferon (IFNgamma) assay to detect infected sheep early in the pathogenesis of Johne's disease enables the removal of such animals from the flock before bacterial shedding and pasture contamination. However, nonspecific IFNgamma responses in the assay have meant that to achieve high-test specificity, there has been a reduction in sensitivity. Although the optimal conditions for the use of the assay in cattle have been well documented, there have been few studies optimizing the assay for use in sheep. The current study details the effect of anticoagulant, duration of incubation, cell concentration, blood storage temperature, time of stimulation of cells with antigen relative to time of sample collection, and temperatures during transit on IFNgamma synthesis. Maximal IFNgamma synthesis occurred with incubation periods of 48 hr in blood collected into heparinized tubes. Decreasing the leukocyte population by diluting the total peripheral blood leukocyte concentration was associated with a decreasing IFNgamma response. Conversely, concentrating the peripheral blood concentration 2-fold resulted in an increase in the IFNgamma production. In field studies, immediate incubation of blood samples with antigen at 37 degrees C resulted in larger IFNgamma responses; however, significantly lower IFNgamma values were obtained if the samples were transported at ambient temperature. The results of this study indicate that optimization of the IFNgamma assay may enable increased synthesis of IFNgamma during the stimulation phase of the assay and that future work may determine whether this translates to increased sensitivity of the assay in detecting early infections in sheep.